Modified glucoamylase enzymes and yeast strains having enhanced bioproduct production

ABSTRACT

The invention is directed to non-natural yeast able to secrete significant amounts of glucoamylase into a fermentation media. The glucoamylase can promote degradation of starch material generating glucose for fermentation to a desired bioproduct, such as ethanol. The glucoamylase can be provided in the form of a glucoamylase fusion protein having a  S. cerevisiae  mating factor alpha 2 (Sc MFα2) or repressible acid phosphatase (Sc PHO5) secretion signal.

CROSS REFERENCE TO RELATED APPLICATION

The present Application claims the benefit of commonly owned U.S. provisional Application having Ser. No. 62/112,807, filed on Feb. 6, 2015, entitled Modified Glucoamylase Enzymes and Yeast Strains Having Enhanced Ethanol Production, which Application is incorporated herein by reference in its entirety. Also, the entire contents of the ASCII text file entitled “CAR0178P1_Sequence_Listing_ST25.txt,” created on Feb. 6, 2015, and having a size of 78 kilobytes is incorporated herein by reference.

FIELD OF THE INVENTION

The current invention relates to modified glucoamylase enzymes, microorganisms expressing these enzymes, and fermentations methods for producing ethanol.

BACKGROUND

Ethanol production by fermentation is a well know industrial process. However increasing ethanol yields can be technically difficult. There are various factors that make it challenging for microorganisms to grow in fermentation conditions designed for increased ethanol production. For example, the fermentation medium may have higher substrate concentrations to promote ethanol production, but these conditions can have a negative impact on cell growth. Also, increased ethanol concentration and accumulation of undesirable byproducts can also be detrimental to cell health. Yeast strains have been selected for tolerance to these conditions, which can result in improved ethanol yields. In particular, the ethanol tolerant strains of the yeast Saccharomyces cerevisiae have been used in industrial settings as a workhorse microorganism for producing ethanol.

Molecular techniques have led to the identification of genes that are associated with ethanol tolerance. For example, Kajiwara (Appl Microbiol Biotechnol. 2000; 53:568-74.) reports that overexpression of the OLE1 gene which is involved in unsaturated fatty acid (UFA) synthesis resulted in higher unsaturated fatty acid levels in the cell and higher ethanol production. Other research has found that accumulation of trehalose by disruption of the trehalose-hydrolyzing enzyme, acid trehalase (ATH) (Kim et al., Appl Environ Microbiol. 1996; 62:1563-1569) or accumulation of proline L-proline by a strain carrying a PRO1 gamma-glutamyl kinase mutation (Takagi, et al., Appl Environ Microbiol. 2005; 71:8656-8662.) improves ethanol tolerance in yeast. Ergosterol is closely associated with ethanol tolerance of Saccharomyces cerevisiae (Inoue, et al., Biosci Biotechnol Biochem. 2000; 64:229-236). While advancements have been made in this area, use of genetically modified strains that demonstrate ethanol tolerance may not alone be sufficient to provide desired levels of ethanol during a fermentation process.

In addition to the genetic profile of the fermentation microorganism, the components of the fermentation medium can have a significant impact on ethanol production. In fermentation processes, a carbohydrate or carbohydrate mixture is present in the medium. Starch is a widely available and inexpensive carbohydrate source. It is available from a wide variety of plant sources such as corn, wheat, rice, barley, and the like. Many organisms are not capable of metabolizing starch directly, or else metabolize it slowly and inefficiently.

Accordingly, it is common to treat starch before feeding it into the fermentation process, in order to break it down into monosaccharides that the organism can ferment easily. Usually, starch is hydrolyzed to form a mixture containing mainly glucose (i.e., dextrose). However, the pre-treatment of a starch composition in preparation for fermentation can be expensive and labor intensive as it commonly involves the addition of purified starch-degrading enzymes to the starch material and requires additional steps prior to carrying out fermentation. Further, complete hydrolysis to glucose adds significant cost, so most commercially available glucose products tend to contain a small amount of various oligomeric polysaccharides.

A significant portion of the cost to produce starch based ethanol is the enzymes that break down the starch into fermentable sugars. Various molecular techniques have been attempted in in Saccharomyces cerevisiae to reduce or eliminate the need to add amylolytic enzymes to the fermentation medium, but these approaches have yielded varying degrees of success. A potential limiting factor affecting the commercial viability of engineered strains is the ability of Saccharomyces cerevisiae to secrete large amounts of foreign protein.

SUMMARY OF THE INVENTION

The invention relates to fermentation methods including non-natural yeast that provide high levels of glucoamylase activity in the fermentation medium. The current invention also relates to glucoamylase enzymes (E.C. 3.2.1.3) that are modified to partially or fully replace their natural secretion sequence with a heterologous secretion sequence. The invention also relates to genes encoding these secretion sequence-modified glucoamylase enzymes, as well as microorganisms expressing these genes. The invention also relates to methods of for producing bio-derived products (fermentation products) manufactured by the organism, such as ethanol. The invention also relates to fermentation coproducts which can be used for other types of compositions, such as animal feed compositions.

In experimental studies associated with the current application, it has been found that the Saccharomyces cerevisiae mating factor alpha 2 (ScMFα2) secretion signal and the Saccharomyces cerevisiae repressible acid phosphatase (ScPHO5) secretion signal, when attached to a glucoamylase enzyme and expressed in microbial cells, can promote high levels of ethanol production during fermentation. Surprisingly, it was found that adding a ScMFα2 secretion signal or ScPHO5 secretion signal to a naturally secreted yeast glucoamylase allowed cells expressing such a modified enzyme to generate substantially more ethanol in the fermentation medium. Some aspects of the invention uses a Saccharomycopsis fibuligera glucoamylase (herein “SfGA”), of an amylolytically active portion thereof, such as a SfGA having 90% or greater sequence identify to amino acids 27-515 of SEQ ID NO:1 for making the Sc MFα2 or Sc PHO5 secretion signal—glucoamylase.

Therefore, aspects of the invention provide a polypeptide comprising (a) a secretion signal amino acid sequence having 90% or greater identity to SEQ ID NO:3 (the Sc MFα2 secretion signal) or SEQ ID NO:5 (the Sc PHO5 secretion signal) and (b) a glucoamylase amino acid sequence from a yeast, fungal, or bacterial glucoamylase polypeptide, wherein the polypeptide has glucoamylase activity. In some aspects the glucoamylase amino acid sequence is based on a glucoamylase sequence from Saccharomycopsis fibuligera. In some aspects, the glucoamylase amino acid sequence has 90% or greater sequence identify to amino acids 27-515 of SEQ ID NO:1, which includes the amylolytically active portion of a SfGA glucoamylase termed “SfGA-1.” In some aspects the polypeptide has 95% or greater sequence identity to SEQ ID NO:7 (Sc MFα2 SS-Sf GA-1) or has 95% or greater sequence identity to SEQ ID NO:9 (Sc PHO5 SS-Sf GA-1)

Aspects of the invention also provide a nucleic acid sequence that encodes a Sc MFα2 secretion signal—glucoamylase enzyme or a Sc PHO5 secretion signal—glucoamylase enzyme. Aspects include nucleic acids having 75% or greater identity to SEQ ID NO:8 (encoding Sc MFα2 SS-Sf GA-1) or having 75% or greater identity to SEQ ID NO:10 (encoding Sc PHO5 SS-Sf GA-1). These aspects include constructs wherein the nucleic acid is present on a vector construct, which may include one or more of the following sequences: a promoter sequence, a terminator sequence, a selectable marker sequence, a genomic integration sequence, and/or a replication origin sequence. The nucleic acid can be integrated into one or more locations of the hosts genomic DNA, or can be present within the cell but not integrated, such as on a plasmid or episomal construct. The invention also provides nucleic acids, such as DNA oligomers (e.g., single stranded DNA PCR primers, or longer linear DNA segments) that can be useful for the detection of the glucoamylase gene with the Sc MFα2 or Sc PHO5 secretion sequence in a cell.

Aspects of the invention also provide a host cell expressing the Sc MFα2 secretion signal—glucoamylase enzyme or a Sc PHO5 secretion signal—glucoamylase enzyme. Is some aspects, the host cell is capable of secreting the enzyme into medium in which the cell is present. The host cell can also be tolerant to a bio-derived product of the cell, such as ethanol or another product, derived from precursors resulting from the amylolytic activity of the enzyme. For example, the host cell can be a commercially available strain or one having one or more specific genetic modifications that provide an increase in tolerance to a bioderived product, such as increased ethanol tolerance. Exemplary host cells include fungal cells such as Saccharomyces cerevisiae.

Aspects of the invention also provide a method for producing a bio-derived product. The method comprises providing a fermentation medium comprising a host cell expressing the Sc MFα2 secretion signal—glucoamylase enzyme or a Sc PHO5 secretion signal—glucoamylase enzyme, wherein a bio-derived product is produced by a fermentation process. The bio-derived product can be one that is derived from enzymatic degradation of a product of a glucose polymer such as starch, amylose, or amylopectin. In an exemplary method, an alcohol (i.e. ethanol) is produced by a fermentation method. In another exemplary method, an organic acid (i.e. lactic acid) is produced by a fermentation method.

Aspects of the invention also provide a method for treating medium comprising a glucose polymer such as starch, amylase, or amylopectin. The method comprises providing medium comprising a glucose polymer and a host cell expressing the Sc MFα2 secretion signal—glucoamylase enzyme or a Sc PHO5 secretion signal—glucoamylase enzyme. In the method, the glucose polymer contacts the glucoamylase enzyme secreted from the cell and the glucose polymer is degraded to glucose. The treated medium can be used for a fermentation process using a different cell, or can be used for a non-fermentation process.

Another aspect of the invention is a fermentation method for producing a fermentation product. The method includes a step of fermenting a liquid medium comprising a starch material and a non-natural yeast comprising an exogenous nucleic acid encoding a polypeptide comprising an glucoamylase. The non-natural yeast provides the medium with an amount of glucoamylase activity of 2.25 U or greater per gram of biomass.

Aspects of the invention also provide a method for producing ethanol by fermentation, wherein the ethanol is present in the fermentation medium at a concentration of 90 g/L or greater. In the method, a liquid medium comprising a starch material and a non-natural yeast comprising a exogenous nucleic acid encoding polypeptide comprising a glucoamylase portion and a signal sequence heterologous to the glucoamylase is fermented. Fermentation can provide an ethanol concentration of about 90 g/L or greater in the liquid medium, such as in the range of about 90 g/L to about 170 g/L.

In another aspect, the invention provides methods and compositions that can be used to prepare feed compositions. The feed compositions include fermentation medium co-products obtained from a fermentation medium derived from the non-natural yeast of the disclosure. For example, after a fermentation process has been completed, some or all of a bioproduct can be removed from the fermentation medium to provide a refined composition comprising non-bioproduct solids. The non-bioproduct solids can include the non-natural yeast, feedstock material in the medium that is not utilized by the yeast, as well as fermentation by-products. The refined composition can be used to form a feed composition, such as a livestock feed composition. The refined composition comprising non-bioproduct solids can provide carbohydrate and protein supplements to improve the nutritional content of a feed composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a picture of a protein gel comparing concentrated extracts from strains of the disclosure.

DETAILED DESCRIPTION

The aspects of the present invention described below are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following detailed description. Rather a purpose of the aspects chosen and described is so that the appreciation and understanding by others skilled in the art of the principles and practices of the present invention can be facilitated.

Aspects of the invention relate to glucoamylase genes that are modified to replace their natural secretion sequence with a heterologous secretion sequence that is either the Saccharomyces cerevisiae mating factor alpha 2 (Sc MFα2) secretion signal or the Saccharomyces cerevisiae repressible acid phosphatase (Sc PHO5) secretion signal. Nucleic acids capable of serving as templates for the expression of these enzymes are also aspects of the invention.

Aspects of the invention also relate to as microorganisms expressing these enzymes, in particular, fungal organisms such as yeast (e.g., Saccharomyces cerevisiae). Such organisms can express a glucoamylase enzyme with either a Sc MFα2 or a Sc PHO5 secretion signal. The glucoamylase enzyme can be secreted from the cell to a fermentation medium where the enzyme can have amylolytic activity on glucose polymers present in the fermentation medium. In turn, the enzyme can cause degradation of the glucose polymers to glucose, which can enter the cell and be used as a carbon source for the production of a target compound, such as ethanol.

The term “exogenous” as used herein, means that a molecule, such as a nucleic acid, or an activity, such as an enzyme activity, is introduced into the host organism. An exogenous nucleic acid can be introduced in to the host organism by well-known techniques and can be maintained external to the hosts chromosomal material (e.g., maintained on a non-integrating vector), or can be integrated into the host's chromosome, such as by a recombination event. An exogenous nucleic acid can encode an enzyme, or portion thereof, that is either homologous or heterologous to the host organism.

The term “heterologous” refers to a molecule or activity that is from a source that is different than the referenced molecule or organism. For example, in the context of the disclosure, a “heterologous signal sequence” refers to a signal sequence that is different from the referenced polypeptide or enzyme. Therefore, when a native signal sequence is removed from a glucoamylase enzyme and replaced with a signal sequence from a different polypeptide, the modified glucoamylase has a “heterologous signal sequence.” Accordingly, a gene or protein that is heterologous to a referenced organism is a gene or protein not found in that organism. For example, a specific glucoamylase gene found in a first fungal species and exogenously introduced into a second fugal species that is the host organism is “heterologous” to the second fungal organism.

Glucoamylases (E.C. 3.2.1.3) are amylolytic enzymes that hydrolyze 1,4-linked a-D-glucosyl residues successively from the nonreducing end of oligo- and polysaccharide chains with the release of D-glucose

Glucoamylases and can also cleave α-1,6 bonds on amylopectin branching points. As used herein, the term “amylolytic activity” with reference to the Sc MFα2 or Sc PHO5 secretion signal—glucocamylase pertains to these enzymatic mechanisms. A glucoamylase polypeptide can be a variant of a naturally occurring glucoamylase, or a portion of a naturally occurring glucoamylase (such as a glucoamylase that is truncated at its N-terminus, its C-terminus, or both), while the glucoamylase polypeptide retains amylolytic activity.

Alternative names for glucoamylases include amyloglucosidase; γ-amylase; lysosomal α-glucosidase; acid maltase; exo-1,4-α-glucosidase; glucose amylase; γ-1,4-glucan glucohydrolase; acid maltase; 1,4-α-D-glucan glucohydrolase.

Most glucoamylases are multidomain enzymes. Many glucoamylases include a starch-binding domain connected to a catalytic domain via an O-glycosylated linker region. The starch-binding domain may fold as an antiparallel beta-barrel and may have two binding sites for starch or beta-cyclodextrin. However, some glucoamylases do not include a starch binding domain (e.g., see Hostinova et al., Archives of Biochemistry and Biophysics, 411:189-195, 2003), or include a non-canonical starch binding domain. For example, the Rhizopus oryzae glucoamylase possesses a C-terminal raw starch binding domain, and the Saccharomycopsis fibuligera IFO 0111 glucoamylase lacks a clear starch binding domain (Hostinova et al., supra). Therefore, some aspects of the invention are directed to glucoamylases that do not include a starch binding domain and that have an N-terminus modified with the Sc MFα2 or Sc PHO5 secretion signal, and other aspects are directed to glucoamylases that include a starch binding domain and that have an N-terminus modified with the Sc MFα2 or Sc PHO5 secretion signal.

Glucoamylases may also have a catalytic domain having a configuration of a configured twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site. Glucoamylases may have a structurally conserved catalytic domain of approximately 450 residues. In some glucoamylases the catalytic domain generally followed by a linker region consisting of between 30 and 80 residues that are connected to a starch binding domain of approximately 100 residues.

Glucoamylase properties may be correlated with their structural features. A structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models (see, e.g., Coutinho, P. M., and Reilly, P. J., 1994. Protein Eng. 7:393-400 and 749-760). It has been shown that the catalytic and starch binding domains are functionally independent based on structure-function relationship studies, and there are structural similarities in microbial glucoamylases. From other studies, specific glucoamylase residues have been shown to be involved in directing protein conformational changes, substrate binding, thermal stability, and catalytic activity (see, for example, Sierks, M. R., et al. 1993. Protein Eng. 6:75-79; and Sierks, M. R., and Svensson, B. 1993. Biochemistry 32:1113-1117). Therefore, the correlation between glucoamylase sequence and protein function is understood in the art, and one of skill could design and express variants of amylolytically active glucoamylases having one or more amino acid deletion(s), substitution(s), and/or additions. For example, in some aspects, the glucoamylase portion of the Sc MFα2 or Sc PHO5 secretion signal—glucoamylase can contain a truncated version of a naturally occurring glucoamylase, the truncated version having, in the least, a catalytic and optionally a starch-binding domain having amylolytic activity as described herein.

Hostinova et al. (Archives of Biochemistry and Biophysics, 411:189-195, 2003) describes the nucleotide sequence of the glucoamylase gene Glm in the yeast strain Saccharomycopsis fibuligera IFO 0111 (referred to herein as “SfGA-1”). According to Hostinova et al., the Saccharomycopsis fibuligera Glm gene is transcribed into a 1.7 kb RNA transcript that codes for a 515 amino acid protein, and is represented by SEQ ID NO:1. In the 515 amino acid-long polypeptide chain 26 N-terminal amino acid residues constitute the signal peptide and subsequent 489 amino acid residues constitute the mature protein. Mature Glm, which lacks the signal sequence and is 489 amino acids long, has a predicted molecular weight of 54,590 Da in deglycosylated form. In an alignment with other glucoamylases, Glm was shown to have homology in the catalytic domain.

Itoh et al. (J. Bacteriol. 169:4171-4176) describes the nucleotide sequence of another glucoamylase gene, GLU1, in the yeast Saccharomycopsis fibuligera (referred to herein as “Sf GA-2”). The Saccharomycopsis fibuligera GLU1 gene is transcribed into a 2.1 kb RNA transcript that codes for a 519 amino acid protein and has a molecular weight of 57,000 Da. GLU1 has four potential glycosylation sites (for asparagine-linked glycosides having a molecular weight of 2000 Da). GLU1 has four potential glycosylation sites (for asparagine-linked glycosides having a molecular weight of 2000 Da). GLU1 has a natural signal sequence for secretion that is cleaved off, likely during export of the protein. The cleaved site is preceded by the basic amino acids Lys-Arg, thought to be a proteolytic processing signal to yield mature protein.

Itoh et al. (supra) also describes alignment of amino acid sequences of glucoamylases from yeast and fungi. Saccharomycopsis fibuligera, A. niger, Rhizopus oryzae, and Saccharomyces diastaticus, and Saccharomyces cerevisiae were aligned showing five highly homologous segments (S1-S5). These parts of the respective conserved segments were shown to be conformationally similar to each other. The S5 segment, generally located at the carboxy termini, appears to be nonessential to amylolytic activities, since glucoamylases from Saccharomyces species lack this region.

In this regard, the invention also contemplates variants and portions of Sf GA having glucoamylase activity. Tables 1 and 2 present a list of various fungal and bacterial glucoamylase genes, including the amino acid location of the native signal sequence, and in some sequences, the propeptide, of the glucoamylase.

TABLE 1 Fungal Glucoamylases Signal Pro- Name Accession Organism peptide peptide Chain GAMP Q03045 Amorphotheca 1-29 30-616 (AMYG_AMORE) resinae (Creosote fungus) (Hormoconis resinae) GLAA P69328 Aspergillus 1-18 19-24 25-640 (AMYG_ASPNG) niger STA1 P04065 Saccharomyces 1-21 22-767 (AMYH_YEASX) cerevisiae STA2 P29760 Saccharomyces 1-21 22-768 (AMYI_YEASX) cerevisiae GLAA P69327 Aspergillus 1-18 19-24 25-640 (AMYG_ASPAW) awamori (Black koji mold) glaA P36914 Aspergillus oryzae 1-19 20-25 26-612 (AMYG_ASPOR) (strain ATCC 42149/ RIB 40) (Yellow koji mold) GAA P42042 Blastobotrys 1-18 19-624 (AMYG_BLAAD) adeninivorans (Yeast) (Arxula adeninivorans) GAM1 P22861 Schwanniomyces 1-22 23-958 (AMYG_SCHOC) occidentalis (Yeast) (Debaryomyces occidentalis) gaI P23176 Aspergillus 1-18 19-24 25-639 (AMYG_ASPKA) kawachii (White koji mold) (Aspergillus awamori var. kawachi) glaA P22832 Aspergillus 1-18 19-24 25-639 (AMYG_ASPSH) shirousami GAM1 O74254 Candida albicans 1-20 21-946 (AMYG_CANAL) (strain SC5314/ ATCC MYA-2876) AMYG_RHIOR P07683 Rhizopus oryzae 1-25 26-604 (Mucormycosis agent) (Rhizopus arrhizus var. delemar) meu17 O60087 Schizosaccharomyces 1-16 17-28 29-450 (mAMYG_SCHPO) pombe (strain 972/ ATCC 24843) (Fission yeast) I2K2N7 Brettanomyces 1-21 22-575 bruxellensis AWRI1499 SGA1 A0A0H5C3I6 Cyberlindnera 1-16 17-577 jadinii (Torula yeast) (Pichia jadinii) GLA1 P26989 Saccharomycopsis 1-27 28-519 (AMYH SACFI) fibuligera (“Sf GA-3”) (Hostinova et al. 2001) GLU1 P08017.1 Saccharomycopsis 1-27 28-519 AMYG_SACFI fibuligera (“Sf GA-2”) (Itoh et al. 1987) Glm CAC83969 Saccharomycopsis 1-26 27-515 (“Sf GA-1”) fibuligera IFO 0111 (Hostinova et al. 2003)

TABLE 2 Bacterial Glucoamylases Signal Pro- Amylase gene Accession Organism peptide peptide Chain SusB G8JZS4 Bacteroides 1-21 22-738 (SUSB_BACTN) thetaiotaomicron (strain ATCC 29148/ DSM 2079/NCTC 10582/E50/VPI-5482) cga P29761 Clostridium sp. 1-21 22-702 (AMYG_CLOS0) (strain G0005)

As noted herein and in Tables 1 and 2, glucoamylases enzymes from various fungal and bacterial species also generally include a native “signal sequence.” Various other terms may be used to indicate a “signal sequence” as known in the art, such as where the word “signal” is replaced with “secretion” or “targeting” or “localization” or “transit” or leader,” and the word “sequence” is replaced with “peptide” or “signal.” Generally, a signal sequence is a short amino acid stretch (typically in the range of 5-30 amino acids in length) that is located at the amino terminus of a newly synthesized protein. Most signal peptides include a basic N-terminal region (n-region), a central hydrophobic region (h-region) and a polar C-terminal region (c-region) (e.g., see von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690). A signal sequence can target the protein to a certain part of the cell, or can target the protein for secretion from the cell. For example, it has been shown that the native N-terminal signal sequence of the S. diastaticus Glucoamylase STAI gene can target it to the endoplasmic reticulum of the secretory apparatus (for example, see Yamashita, I. et al., (1985) J. Bacteriol. 161, 567-573).

In one aspect, the current invention provides the partial or full replacement of the native signal sequence of a glucoamylase enzyme with the Sc MFα2 or Sc PHO5 secretion signal. In another aspect, the current invention provides addition of the Sc MFα2 or Sc PHO5 secretion signal to a glucoamylase enzyme, in addition to its native secretion signal. In view of the addition of the heterologous secretion signal, the proteins may be referred to as “fusion proteins,” and annotated as follows: [Sc MFα2-SS]-[GA] and [Sc PHO5-SS]-[GA].

The Saccharomyces cerevisiae mating factor alpha 2 (Sc MFα2) secretion signal is described in U.S. Pat. No. 4,546,082 (Kurjan et al.). The Sc MFα2 SS sequence is as follows: MKFISTFLTFILAAVSVTA (SEQ ID NO. 10). The Sc MFα2 sequence is from the gene YGL089C (YGL089C), whereas MFα1 is coded by the gene YPL187W MFα1 and MFα2 are pheromones secreted by MATa cells. In one aspect, a glucoamylase fusion protein comprises a secretion signal sequence that has 90% or greater identity to SEQ ID NO: 10. For example, one amino acid of SEQ ID NO: 10 can be substituted with an amino acid, such as a conservative amino acid.

The Saccharomyces cerevisiae repressible acid phosphatase (Sc PHO5) secretion signal is described in U.S. Pat. No 5,521,086 (Scott et al.) and Meyhack et al. (EMBO J. 6:675-680, 1982). The Sc PHO5 SS sequence is as follows: MFKSVVYSILAASLANA (SEQ ID NO. 11). The Sc PHO5 sequence is from PHO5 which is a structural gene that encodes a S. cerevisiae acid phosphatase, which is regulated by the concentration or inorganic phosphate (P_(i)) in the medium. In one aspect, a glucoamylase fusion protein comprises a secretion signal sequence that has 90% or greater identity to SEQ ID NO. 11. For example, one amino acid of SEQ ID NO. 11 can be substituted with an amino acid, such as a conservative amino acid.

Molecular techniques can be performed to create a nucleic acid sequence that is a template for the expression of the Sc MFα2 SS or the Sc PHO5 SS-glucoamylase gene (if the glucoamylase protein/nucleotide sequences are known in the art). As a general matter, a nucleic acid is prepared to encode a protein comprising the Sc PHO5 SS- or Sc MFα2 SS sequence and a glucoamylase sequence.

Any sequence encoding a functional glucoamylase polypeptide can be used. In some aspects, the glucoamylase sequence can be a native (“wild type”) sequence of a glucoamylase gene, where the sequence of the glucoamylase portion of the Sc MFα2 SS or the Sc PHO5 SS-glucoamylase gene does not differ from the native sequence at any amino acid position. In other aspects, the sequence of the glucoamylase portion of the Sc MFα2 SS or the Sc PHO5 SS-glucoamylase gene differs from the native sequence at one or more amino acid position(s). The difference can be, for example, (a) the removal of one or more amino acids from the wild type sequence, (b) the addition of one or more amino acids to the wild type sequence, (c) the substitution of the wild type sequence, a combination of (a) and (c), or a combination of (b) and (c).

For example, in one aspect the native sequence of the glucoamylase can be altered at its N-terminus prior to adding the Sc MFα2 SS or the Sc PHO5 SS sequence. In some aspects, all or a portion of the native glucoamylase signal sequence is removed prior to attaching the Sc MFα2 SS or the Sc PHO5 SS sequence. For example, a portion of a native leader sequence of the glucoamylase can be altered by deletion of one or more, but not all, amino acids of the native secretion signal (e.g., deletion of up to 50%, 60%, 70%, 80, 90%, or 95% of the native leader sequence). Such deletion of a portion of the native leader sequence may cause the native glucoamylase leader to lose its native functionality, which is replaced with the functionality provided by the Sc MFα2 or Sc PHO5 secretion signal. In other aspects, all of the native secretion signal can be removed from the glucoamylase and replaced with the Sc MFα2 SS or the Sc PHO5 SS sequence.

For example, and with reference to Table 1, in preparing a fusion protein construct the first 18 amino acids of the S. fibuligera IFO 0111 glucoamylase (Sf GA-1), which corresponds to the predicted leader sequence using the CBS prediction server (i.e., amino acids 1-18 of SEQ ID NO:12), is removed. Therefore, a portion of the S. fibuligera glucoamylase native secretion signal is replaced with the Sc MFα2 SS sequence (SEQ ID NO:10; 19 amino acids) or with the Sc PHO5 SS sequence (SEQ ID NO:11; 17 amino acids) which can then be attached directly or indirectly to the remaining portion of the S. fibuligera glucoamylase polypeptide (e.g., amino acids 19-515 of SEQ ID NO:1). This provides a Sc MFα2 SS-Sf GA of 516 amino acids (SEQ ID NO:13) or a Sc PHOS SS-Sf GA of 514 amino acids (SEQ ID NO:14).

As another example, one or more amino acids of a native leader sequence of the glucoamylase can be altered by substitution, which is the replacement of the native amino acid at a particular location in the native glucoamylase leader with an amino acid that is different than the native amino acid. For example, a portion of a native leader sequence of the glucoamylase can be altered by substitution of one or more amino acids of the native secretion signal (e.g., up to 50%, 60%, 70%, 80%, 90%, or 95% of the native leader sequence amino acids can be substituted). Substitution of one or more amino acids may cause the native glucoamylase leader to lose its native functionality, which is replaced with the functionality provided by the Sc MFα2 or Sc PHO5 secretion signal.

In other aspects, the fusion polypeptide comprising the Sc PHOS SS- or Sc MFα2 SS sequence and a glucoamylase sequence optionally comprises additional sequence that is not present in the native glucoamylase polypeptide, or either Sc PHO5 SS- or Sc MFα2 SS sequence. The additional sequence can provide functionality to the secretion signal-modified glucoamylase that is not present in the native polypeptide. Additional functionalities include, for example, protease sites or binding sites for other proteins or materials.

An example of an additional sequence that may not be present in the native glucoamylase polypeptide, or either the Sc PHO5 SS- or Sc MFα2 SS sequences, but that can be added is a linker or spacer sequence. A linker sequence can be located between the Sc PHO5 SS- or Sc MFα2 SS sequence and the glucoamylase sequence. Such fusion polypeptides [secretion signal modified polypeptide] can be annotated as follows: [Sc MFα2-SS]-[L]-[GA] and [Sc PHO5-SS]-[L]-[GA], wherein “L” denotes one or more amino acids that link the signal sequence to the glucoamylase. Exemplary linkers include up to 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. A linker can include amino acids that cause the linker to be rigid and prevent interactions between the secretion signal and other portions of the glucoamylase. Rigid linkers may include residues such as Pro, Arg, Phe, Thr, Glu, and Gln. Alternatively, the fusion polypeptide can include a flexible linker. Flexible linkers can include glycine residues and connect the signal sequence to the glucoamylase portion of the fusion protein without interfering with their respective functions. In some aspects the polypeptide includes a linker having a protease cleavage sequence. Exemplary protease cleavage sequences include those for thrombin, factor Xa, rhinovirus 3C, TEV protease, Ssp DnaB, intein, Sce VMA1 intein, enterokinase, and KEX2 (see, for example, Waugh, D. S., Protein Expr Purif. 80 (2): 283-293, 2011; Zhou et al., Microbial Cell Factories 13:44, 2014; and Bourbonnais et al., J. Bio. Chem. 263(30):15342, 1988)

Another example of an additional sequence that may not be present in the native glucoamylase polypeptide, or either the Sc PHOS SS- or Sc MFα2 SS sequences, but that can be added, is a tag sequence. A tag sequence can be located at the C-terminus of the glucoamylase sequence, and such proteins can be annotated as follows: [Sc MFα2-SS]-[GA]-[T] and [Sc PHO5-SS]-[GA]-[T], wherein “T” denotes one or more amino acids that provide the tag sequence. Exemplary peptide tags include up to 5, 10, 15, or 20 amino acids. The peptide tag can be useful for any one or more of a variety of purposes. For example, the tag can allow purification of the enzyme from the medium by the ability of a tag-binding member to specifically interact with the tag. The tag can also allow detection or identification of the protein using a tag-binding member with a detectable label. Exemplary short peptide tags are poly-Arg, FLAG, poly-His, c-myc, S, and Strep II.

Secretion signal modified polypeptides of the disclosure can also have deletions to one or more regions of the native glucoamylase polypeptide other than the native secretion sequence, wherein the deletions do not affect the polypeptides' amylolytic activity. The deletions can be based on known information regarding the structure and function of native glucoamylases, including mutational studies and sequence alignments (e.g., see Coutinho, supra, and Sierks, supra.). In some aspects the secretion signal modified polypeptides have up to 1%, up to 2%, up to 4%, up to 6%, up to 8%, up to 10%, up to 12%, up to 14%, up to 16%, up to 18%, up to 20%, or up to 25% of the glucoamylase polypeptide's sequence is deleted. In some aspects, the secretion signal modified polypeptides of the disclosure have a deletion of a portion of the C-terminus corresponding to the native glucoamylase polypeptide.

Truncated forms of glucoamylase have been generated and have been shown to have enzymatic activity. For example Evans et al. (Gene, 91:131; 1990) generated a series of truncated forms of glucoamylase to investigate how much of the O-glycosylated region was necessary for the activity or stability of GAII, a fully active form of the enzyme lacking the raw starch-binding domain. It was found that a significant portion of the C-terminus could be deleted from GAII with insignificant effect on activity, thermal stability, or secretion of the enzyme.

Various amino acids substitutions associated with causing a change in glucoamylase activity are also known in the art. Substitution(s) of amino acid(s) at various locations in the glucoamylase sequence have been shown to affect properties such as thermo stability, starch hydrolysis activity, substrate usage, and protease resistance. As such, the current disclosure contemplates use of the Sc MFα2 or Sc PHO5 secretion signal with a glucoamylase sequence that includes one or more amino acids substitution(s) in the glucoamylase portion of the polypeptide, wherein the substitutions differ from the wild type sequence of the glucoamylase.

For example, U.S. Pat. No. 8,809,023 describes a method for reducing the ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) during the hydrolysis of starch. In particular a Trichoderma reesei glucoamylase (Tr GA) is described (total length of 632 amino acids having an N-terminal having a signal peptide) that is modified at with amino acid positions as follows: D44R and A539R; or D44R, N61I, and A539R. This glucoamylase variant is reported to exhibit a reduced IS/SH ratio compared to said parent glucoamylase during the hydrolysis of starch. As an example, the current disclosure contemplates the replacement of the native leader sequence of a desired glucoamylase (e.g., Sf GA with the Sc MFα2 or Sc PHO5 secretion signal, wherein the desired glucoamylase further has amino acid substitutions corresponding to the D44R and A539R; or D44R, N61I and A539R substitutions of the modified Tr GA. In a broader sense, the Sc MFα2 or Sc PHO5 secretion signal could be used with a glucoamylase variant having amino acid substitutions: D44R and A539R; or D44R, N61I and A539R, the positions corresponding to the respective position in the TrGA sequence, wherein said glucoamylase variant has at least 90% amino acid sequence identity to the entire length of the TrGA sequence. The corresponding “respective position” of a template glucoamylase sequence to the TrGA sequence can be understood by a sequence alignment of, for example, known glucoamylase polypeptide sequences (the template for construction of a Sc PHO5 SS- or Sc MFα2 SS glucoamylase fustion), to the TrGA sequence.

As another example, U.S. Pat. No. 8,592,194 describes glucoamylase variants with increased thermo stability compared to wild type glucoamylase variants. Also described in this disclosure is the Trichoderma reesei glucoamylase but instead one or more amino acid substitutions to the native Tr GA sequence at positions 10, 14, 15, 23, 42, 45, 46, 59, 60, 61, 67, 68, 72, 73, 97, 98, 99, 102, 108, 110, 113, 114, 122, 124, 125, 133, 140, 144, 145, 147, 152, 153, 164, 175, 182, 204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 240, 241, 242, 244, 263, 264, 265, 268, 269, 276, 284, 291, 300, 301, 303, 310, 311, 313, 316, 338, 342, 344, 346, 349, 359, 361, 364, 379, 382, 390, 391, 393, 394, 408, 410, 415, 417, and 418. As an example, the current disclosure contemplates the replacement of the native leader sequence of Sf GA with the Sc MFα2 or Sc PHO5 secretion signal, wherein the Sf GA further has any one or more of the amino acid substitutions that are demonstrated in providing increased thermostability. In a broader sense, the Sc MFα2 or Sc PHOS secretion signal could be used with a glucoamylase variant having amino acid substitutions providing increased thermostability, the positions corresponding to the respective position in the TrGA sequence.

The determination of “corresponding” amino acids from two or more glucoamylases can be determined by alignments of all or portions of their amino acid sequences. Sequence alignment and generation of sequence identity include global alignments and local alignments, which typically use computational approaches. In order to provide global alignment, global optimization forcing sequence alignment spanning the entire length of all query sequences is used. By comparison, in local alignment, shorter regions of similarity within long sequences are identified.

As used herein, an “equivalent position” means a position that is common to the two sequences (e.g., a Sf GA sequence and a GA sequence having the desired substitution(s)) that is based on an alignment of the amino acid sequences of one glucoamylase or as alignment of the three-dimensional structures. Thus either sequence alignment or structural alignment, or both, may be used to determine equivalence.

In some modes of practice, the BLAST algorithm is used to compare and determine sequence similarity or identity. In addition, the presence or significance of gaps in the sequence which can be assigned a weight or score can be determined. These algorithms can also be used for determining nucleotide sequence similarity or identity. Parameters for to determine relatedness are computed based on art known methods for calculating statistical similarity and the significance of the match determined. Gene products that are related are expected to have a high similarity, such as greater than 50% sequence identity. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as follows

In some modes of practice, an alignment is performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.29 software with default parameters. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the BLAST version 2.2.29 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence. A global alignment can align sequences with significant identity to, for example, the S. fibuligera glucoamylase in order to determine which corresponding amino acid position(s) in the target sequence (e.g., a glucoamylase ortholog) can be substituted with the one or more of the amino acid if a glucoamylase variant is used.

Nucleic acids sequences encoding the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase polypeptide, as well as any regulatory sequence (e.g., terminator, promoter, etc.) and vector sequence (e.g., including a selection marker, integration marker, replication sequence, etc.) can, in some modes of practice, be prepared using known molecular techniques. General guidance for methods for preparing DNA constructs (e.g., for the DNA constructs including the Sc MFα2 SS or the Sc PHO5 SS-glucoamylase gene) can be found in Sambrook et al Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

When small amounts of glucoamylase template DNA are used as starting material in PCR, primers that include the MFα2 SS or the Sc PHO5 SS sequences and a portion of the glucoamylase sequence that is 3′ to its native signal sequence can be used to generate relatively large quantities of a specific DNA fragment that includes the MFα2 SS or the Sc PHO5 SS sequences and the glucoamylase gene.

PCR techniques can be used for modifying a native glucoamylase nucleic acid sequence to add the Sc MFα2 SS or the Sc PHO5 SS sequences, or to introduce one or more mutations in the glucoamylase nucleic acid sequence to provide a variant. PCR techniques are described in, for example, Higuchi, (1990) in PCR Protocols, pp. 177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al (1989) Nuc. Acids Res. 17:723-733. The techniques may optionally include site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding a glucoamylase polypeptide.

Alternatively, nucleic acid molecules can be generated by custom gene synthesis providers such as DNA2.0 (Menlo Park, Calif.) or GeneArt (Life Technologies, Thermo Fisher Scientific).

An expression vector can be constructed to include the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase nucleic acid sequence operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the host organisms include, for example, plasmids, episomes and artificial chromosomes. The vectors can include selection sequences or markers operable for stable integration into a host chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.

In some aspects, the nucleic acid can be codon optimized. The nucleic acid template that is used for the glucoamylase portion of the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase can be the native DNA sequence that codes for the glucoamylase, or the template can be a codon-optimized version that is optimized for expression in a desired host cell. Databases that provide information on desired codon uses in particular host organisms are known in the art.

According to one aspect of the disclosure, a DNA construct comprising a Sc MFα2 SS or the Sc PHO5 SS—glucoamylase is operably linked to a promoter sequence, wherein the promoter sequence is functional in a host cell of choice. In some aspects, the promoter shows transcriptional activity in a fungal host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. In some aspects the promoter is useful for expression in S. cerevisaie. Examples of well-known constitutive promoters include, but are not limited to the cytochrome c promoter (pCYC), translational elongation factor promoter (pTEF), the glyceraldehyde-3-phosphate dehydrogenase promoter (pGPD), the phosphoglycerate kinase promoter (PGK), and the alcohol dehydrogenase promoter (pADH). Optionally, an additional factor that controls expression such as an enhancer or the like may also be included on the vector.

The expression vector including the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene can also include any termination sequence functional in the host cell. For example, the termination sequence and the promoter sequence can be from the same cell, or the termination sequence is homologous to the host cell. The termination sequence can correspond to any promoter that is used.

The DNA construct may be introduced into a host cell using a vector. The vector may be any vector which when introduced into a host cell is stably introduced. In some aspects, the vector is integrated into the host cell genome and is replicated. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like. In some aspects, the vector is an expression vector that comprises regulatory sequences operably linked to the glucoamylase coding sequence.

The DNA construct comprising a Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene can further include a selectable marker, thereby facilitating the selection in a host cell. For example, the selectable marker can be for transformed yeast. Examples of yeast selectable marker include markers commonly used for selecting for transformed yeast cells. Auxotrophic markers can be used using a gene that controls an auxotrophy, meaning that the gene enables yeast to produce a nutrient required for the growth of the yeast. Examples genes that control auxotrophies include leucine auxotrophy (LEU2), histidine auxotrophy (HIS3), uracil auxotrophy (URA3, URA5), and tryptophan auxotrophy (TRP1).

The DNA construct may be one which is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. For example, a fungal cell may be transformed with the DNA construct encoding the glucoamylase, and integrating the DNA construct, in one or more copies, in the host chromosome(s). This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, such as by homologous or heterologous recombination.

The non-natural yeast can also include one or other genetic modifications that are different than the modification of the glucoamylase with heterologous signal sequence. For example, one or more additional modifications can include those that provide a different polysaccharide-degrading enzyme, such as an exogenous or modified alpha-amylase, beta-amylase, pullulanase, isoamylase, or cyclodextrin glycosyltransferases; an exogenous or modified sugar transporter gene (such as an isomaltose transporter); and/or an exogenous or modified gene that converts a low molecular weight non-glucose sugar to glucose, such as an isomaltase.

Various host cells can be transformed with a nucleic acid including the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene. In some aspects the nucleic acid including the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene is present in a bacterial cell. The bacterial cell can be used, for example, for propagation of the nucleic acid sequence or for production of quantities of the polypeptide.

In other aspects, the host cell is a eukaryotic cell, such as a fungal cell.

In some aspects the host cell is has tolerance to a higher amount of a bioderived product, such as ethanol, in the fermentation medium. In some aspects, the host cell is an “industrial yeast” which refers to any yeasts used conventionally in ethanol fermentation. Examples include sake yeasts, shochu yeasts, wine yeasts, beer yeasts, baker's yeasts, and the like. Sake yeasts demonstrate high ethanol fermentability and high ethanol resistance and genetic stability. Typically, an industrial yeast has high ethanol resistance and preferably is viable at ethanol concentrations of 10% or greater.

In exemplary aspects, the host cell is S. cerevisiae. Some S. cerevisiae have high tolerance to ethanol. Various strains of ethanol tolerent yeast are commercially available, such as RED STAR® and ETHANOL RED® yeast (Fermentis/Lesaffre, USA), FALI (Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC® yeast (Ethanol Technology, Wis., USA), BIOFERM AFT and XR (NABC—North American Bioproducts Corporation, GA, USA), GERT STRAND (Gert Strand AB, Sweden), and FERMIOL (DSM Specialties).

Industrial yeasts are typically prototrophic and therefore do not have an auxotrophic marker suitable for selecting for a transformant. If the host cell does not have the genetic background that would otherwise facilitate retention of the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene within the cell upon transformation, the host cell can be engineered to introduce one or more genetic mutation(s) to establish use of a marker gene in association with and to maintain the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene in the cell. For example, a commercially available ethanol tolerant yeast cell can be genetically modified prior to introducing the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene in the cell.

A marker for a different auxotrophy can be provided by disrupting the gene that controls the auxotrophy. In one mode of practice, an ethanol tolerant strain of yeast is engineered to disrupt copies of one or more genes that control auxotrophies, such as LEU2, HIS3, URA3, URA5, and TRP1. In the case of providing uracil auxotrophy, for example, a normal ura3 gene of an ethanol tolerant yeast can be replaced with an ura3⁻ fragment obtained from a uracil auxotrophic mutant (for example, a Saccharomyces cervisiae MT-8 strain) to disrupt the normal ura3 gene. In the case of a ura3 gene-disrupted strain, the presence/absence of a marker can be easily identified or selected by taking advantage of the fact that a ura3 gene-disrupted strain is able to grow in a medium containing 5-fluoroorotic acid (5-FOA) while a normal ura3 strain (wild-type yeast or usual industrial yeast) is not able to grow. In the case of a lys2 gene-disrupted strain, the presence/absence of a marker can be easily identified or selected by taking advantage of the fact that a lys2 gene-disrupted strain is able to grow in a medium containing α-aminoadipic acid while a normal lys2 strain (wild-type yeast or usual industrial yeast) is not able to grow. Methods for disrupting an auxotrophy-controlling gene and for selectively separating auxotrophy-controlling gene mutants may be used depending on the auxotrophy employed. Alternatively, one can employ dominant selection markers, such as the amdS from Aspergillus nidulans (U.S. Pat. No. 5,876,988), which allows for growth on acetamide as the sole nitrogen source; or ARO4-OFP, which allows for growth in the presence of fluoro-phenylalanine (Fukuda et. al.). These markers can be used repeatedly using the recyclable cre-loxP system, or alternatively can be used to create auxotrophic strains that allow additional markers to be utilized.

After the host cell has been engineered to provide a desired genetic background for introduction of the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene, the gene construct is introduced into a cell to allow for expression. Methods for introducing a gene construct into a host cell include transformation, transduction, transfection, co-transfection, electroporation. In particular, yeast transformation can be carried out suing the lithium acetate method, the protoplast method, and the like. The gene construct to be introduced may be incorporated into a chromosome in the form of a plasmid, or by insertion into the gene of a host, or through homologous recombination with the gene of a host. The transformed yeast into which the gene construct has been introduced can be selected with a selectable marker (for example, an auxotrophic marker as mentioned above). Further confirmation can be made by measuring the activity of the expressed protein.

The transformation of exogenous nucleic acid sequences including the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

The non-natural yeast of the disclosure can be provided in any suitable form. In some aspects, the non-natural yeast is dehydrated to form a dry yeast composition. The dry yeast composition can have increased shelf life over wet compositions.

Fermentation using a host cell expressing the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene can be performed in the presence of a starch and/or sugar containing plant material, referring to a starch and/or sugar containing plant material derivable from any plant and plant part, such as tubers, roots, stems, leaves and seeds. Starch and/or sugar comprising plant material can be obtained from cereal, such as barley, wheat, maize, rye, sorghum, millet, barley, potatoes, cassava, or rice, and any combination thereof. The starch-and/or sugar comprising plant material can be processed, such as by methods such as milling, malting, or partially malting. In some aspects, the starch material is from corn flour, milled corn endosperm, sorghum flour, soybean flour, wheat flour, biomass derived starch, barley flour, and combinations thereof.

In some aspects, the fermentation medium includes a treated starch. For example, the fermentation medium can include a partially hydrolyzed starch. The partially hydrolyzed starch can include high molecular weight dextrins and high molecular weight maltodextrins. In some modes of practice, a partially hydrolyzed starch product having a dextrose equivalent (“DE”) in the range of about 5 to about 95 or more preferably about 45 to about 65, is used in the fermentation medium. Partially hydrolyzed starches and preparation thereof are well known in the art. Partially hydrolyzed starches can be prepared by heating the starch with an acid such as hydrochloric or sulfuric acid at a high temperature and then neutralizing the hydrolysis mixture with a suitable base such as sodium carbonate. Alternatively, partially hydrolyzed starches can be prepared by an enzymatic process, such as by adding alpha-amylase to a starch preparation. An alpha amylase can cause the endohydrolysis of (1→4)-alpha-D-glucosidic linkages in polysaccharides containing three or more (1→4)-alpha-linked D-glucose units. A partially hydrolyzed starch product can be used that have amounts of starch and starch degradation products within desired ranges.

In aspects of the disclosure, given production and secretion of the glucoamylase from the engineered yeast into the fermentation medium, the fermentation method may omit addition of purified or enriched commercial glucoamylase into the medium, or at least allow significantly less commercial glucoamylase to be used in a fermentation method. For example, the engineered yeast of the disclosure can allow addition of commercial glucoamylase to be eliminated or at least reduced by about 50%, 60%, 70%, 80%, 90%, or 95%. Typically amounts of glucoamylase in the range of about 7 units to about 50 units per liter would be used in fermentation methods that do not use a glucoamylase-secreting engineered yeast. The fermentation broth includes water and preferably includes nutrients, such as a nitrogen source (such as proteins), vitamins and salts. A buffering agent can also be present in the fermentation medium. Other components may also be present in the fermentation broth after a period of fermentation, such as fermentation products which can accumulate as the fermentation progresses, and other metabolites. Optionally, the fermentation broth can be buffered with a base such as calcium hydroxide or calcium carbonate, ammonia or ammonium hydroxide, sodium hydroxide, or potassium hydroxide in order to maintain a pH at which the organism functions well.

The engineered yeast of the current disclosure can also be described in terms of the engineered yeast's specific growth rate. Specific growth rate is measured in units of hours⁻¹ measured using techniques known in the art.

The fermentation is carried out under conditions so that fermentation can occur. Although conditions can vary depending on the particular organism and desired fermentation product, typical conditions include a temperature of about 20° C. or greater, and more typically in the range of about 30° C. to about 50° C. During fermentation the reaction mixture can be mixed or agitated. In some modes of practice, the mixing or agitation can occur by the mechanical action of sparging gas to the fermentation broth. Alternatively direct mechanical agitation such as by an impellor or by other means can be used during fermentation.

The non-natural yeast can have an increased tolerance to growth at temperatures that greater than those in which yeast, such Saccharomyces cerevisiae, typically grow at. For example, S. cerevisiae typically have optimal growth in the temperature range of 30° C.-33° C. In some aspects, the non-natural yeast of the disclosure display improved tolerance to growth at temperatures in the range of 34° C.-40° C.

For example, as compared to reference yeast without the genetic modification, the non-natural yeast of the disclosure can have a specific growth rate at a temperature in the range of 34° C.-40° C., that is 10%, 20%, 30%, 40%, or 50% greater than the growth rate of a reference yeast without the genetic modification.

In some cases fermentation is carried out in industrial capacity fermenters in order to achieve commercial scale economic benefits and control. In an aspect, the fermentation is carried out in a fermenter that has a capacity of about 10,000 liters or more.

The pH of the fermentation medium can be adjusted to provide optimal conditions for glucoamylase activity, cell growth, and fermentation activity to provide a desired product, such as ethanol. For example, pH of the solution can be adjusted to in the range of 3 to 5.5. In one mode of practice, the pH of the fermentation medium is in the range of 4 to 4.5.

As noted above, the present fermentation process using genetically modified microorganisms expressing the Sc MFα2 SS or the Sc PHO5 SS—glucoamylase gene and capable of secreting the enzyme produced into the fermentation medium. These enzymes are therefore directly exposed to the broth conditions and affect the carbohydrate composition in the fermentation medium. In the fermentation medium the glucoamylase can cause hydrolysis and release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharide molecules by cleaving alpha-(1,4) and alpha-(1,6) glucosidic bonds.

Starch may also be acted on by one or more other amylases (e.g., alpha-amylase) present in the fermentation medium. For example, if alpha-amylase is present in the fermentation medium it can cause partial hydrolysis of precursor starch and cause a partial breakdown of the starch molecules by hydrolyzing internal alpha-(1,4)-linkages.

In some modes of practice, the fermentation is carried out as a single batch until completion.

In other modes of practice, the fermentation is carried out as a fed batch fermentation process. In this mode of practice, a first portion of a total amount of starch material to be fermented is added to the fermentation medium wherein the glucoamylase enzyme acts on the starch to cause formation of glucose to be used as a substrate for fermentation. Additional starch material is added in one or more portions to provide more substrate for the glucoamylase enzyme in the medium. The addition of starch can be regulated and the formation of glucose can be monitored to provide efficient fermentation.

Preferably, the fermentation is carried out in a continuous mode of operation. In this mode, multiple fermenters operate in series in which a starch hydrolysate is supplied in the first fermenter, which is fed to second fermenter and so on until the starch hydrolysate is converted to ethanol. Continuous operation can be operated using between 2-7 fermenters.

In some modes of practice, a portion of the total amount of starch material is added to the fermentation broth using a variable rate addition system. Examples of such systems include a variable speed pump or a metering valve (such as a throttle valve) operably connected to a pump, which pump or valve can be utilized to vary the amount of starch material introduced into the fermentation broth over time. In an some modes of practice, during the addition of a portion of the starch material, glucose concentration is monitored by a real-time monitoring system.

Real-time monitoring systems include systems that directly monitor glucose concentration and systems that indirectly monitor glucose concentration. Examples of real-time monitoring systems that typically directly monitor glucose concentration include systems based on infrared (IR) spectroscopy, near-infrared (NIR) spectroscopy systems, Fourier transform infrared (FTIR) systems, systems based on refractive index, automated enzyme based measurement systems such as a YSI 2950 Biochemistry Analyzer sold by YSI Life Sciences systems, high performance liquid chromatography (HPLC) based systems, gas chromatography (GC) based systems, and other real-time monitoring systems known to one of skill in the art. Additionally real-time monitoring systems that indirectly monitor/measure the glucose concentration of a fermentation process can be developed by determining the typical carbon distribution in a particular fermentation process and correlating the glucose concentration present in the fermentation broth to another parameter exhibited by the fermentation, such as, for example, a correlation of the glucose level present in the fermentation broth with a measurement of the carbon dioxide evolution rate and the amount of carbon dioxide present in an off-gas stream from the fermentation vessel. The carbon dioxide can be readily measured through use of a mass spectrometer or other suitable instrumental technique for measuring the components of the off-gas stream. In a preferred aspect, the glucose concentration is monitored by a real-time monitoring system using infrared spectroscopy. In another one aspect, the glucose concentration is monitored by a real-time monitoring system using near-infrared spectroscopy. The real time monitoring systems interface with equipment that controls the introduction of starch material into the fermentation broth to modulate the formation of glucose to a desired concentration in the fermentation broth.

During the fermentation process a sample of the fermentation medium can be taken to determine the amount of glucoamylase activity in the medium. The amount of glucoamylase activity in the medium can be referred to as extracellular glucoamylase activity as it corresponds to glucoamylase secreted from the engineered yeast. In some modes of measuring, the amount of glucoamylase activity in the medium can be determined by the amount of glucoamylase activity per amount of biomass per volume of medium.

As used herein “biomass” refers to the weight of the engineered yeast, which can be measured in grams of dried cell weight per liter of medium (DCW/L).

A unit (U) of GA activity can be defined as the amount of enzyme that catalyzes the release of 1 mg glucose/min from starch. Glucoamylase activity can be measured in concentrated broth by coupling starch hydrolysis to a HXK/G6PDH reaction mix (Sigma G3293) in a two-step end point assay. Broth can be concentrated from a predetermined amount of cells grown using a non-glucose carbon source (i.e. raffinose) to avoid interference with the assay.

The specific activity is equal to the activity in a given volume of broth divided by the wet weight of cells in the same volume of broth. Specific activity has the following units, U of GA activity per gram of biomass (U/g biomass). The amount of biomass used in the assay can be measured by determining the wet cell weight after removing the broth, either by filtration or centrifugation.

A starch solution is prepared by dissolving 1.1 g of corn starch (S4126, Sigma) in 50 mL of near boiling water, then adding 1 mL of 3M sodium acetate pH 5.2. A volume of concentrated broth (V_(b)), typically in the range of 1-20 ul (prepared by using a 10 Kb Kd cutoff column, Millipore #UFC901008) is added to the starch slurry (V_(s)), in a total volume of 200 ul, and allowed to incubate at 37° C. for a specific period of time (T), typically between 5-60 minutes. Parameters are selected such that the glucose formation is linear within a desired time. 20 μL of each sample is added to 2 μL 0.6 N NaOH and mixed well. 200 μL of the HXK/G6PDH mix is then added and incubated at 30° C. for 30 minutes. The absorbance at 340 nm is measured using a spectrophotometer (SpectraMax™ M2). Regression analysis using known glucose standards is used to calculate the amount of glucose released in each sample. The specific enzyme activity per gram of biomass (U/g biomass) can be calculated by obtaining the weight in grams of the sample used prior to concentration. Unit of activity=(mg glucose/T)*((V_(b)+V_(s))/(V_(b)))*(222/20). Specific activity=Unit of activity/g biomass.

In some aspects, in the fermentation method the medium has an amount of glucoamylase activity of 2.25 U or greater per gram of biomass. In some aspects the medium has an amount of glucoamylase activity of about 2.3 U or greater, about 2.35 U or greater, about 2.4 U or greater, about 2.45 U or greater, about 2.5 U or greater, about 2.6 U or greater, about 2.7 U or greater, about 2.8 U or greater, about 2.9 U or greater, about 3 U or greater, about 3.5 U or greater, about 4 U or greater, about 4.5 U or greater, about 5 U or greater, about 5.5 U or greater, about 6 U or greater, about 6.5 U or greater, about 7 U or greater, about 7.5 U or greater, or about 8 U or greater per gram of biomass. In some aspects the medium has an amount of glucoamylase activity in the range of about 2.3 U to about 15 U, about 2.4 U to about 15 U, about 2.5 U to about 15 U, about 3 U to about 15 U, about 3.5 U to about 15 U, about 4 U to about 15 U, about 4.5 U to about 15 U, about 5 U to about 15 U, about 5.5 U to about 15 U, about 6 U to about 15 U, about 6.5 U to about 15 U, about 7 U to about 15 U, about 7.5 U to about 15 U, or about 8 U to about 15 U per gram of biomass.

In other aspects, an amount of glucoamylase activity in a fermentation medium provided by a non-natural yeast of the disclosure can be described relative to a reference yeast. For example, the amount of glucoamylase activity that a non-natural yeast expressing an exogenous glucoamylase having a heterologous signal sequence (e.g., having 90% or greater identity to SEQ ID NO:10 or SEQ ID NO:11) can be compared to an otherwise identical yeast expressing the exogenous glucoamylase with its native signal sequence.

In some aspects, the non-natural yeast expressing an exogenous glucoamylase having a heterologous signal sequence provides an amount of glucoamylase activity in the fermentation medium that is at least 1.125 times greater (12.5% greater) than a reference yeast. In some aspects the amount of glucoamylase activity is at least 1.15 times greater, at least 1.175 times greater, at least 1.225 times greater, at least 1.25 times greater, at least 1.3 times greater, at least 1.35 times greater, at least 1.4 times greater, at least 1.45 times greater, at least 1.5 times greater, at least 1.75 times greater, at least 2 times greater, at least 2.25 times greater, at least 2.5 times greater, at least 2.75 times greater, at least 3 times greater, at least 3.25 times greater, at least 3.5 times greater, at least 3.75 times greater, or at least 4 times greater in the non-natural yeast over the reference yeast. In some aspects the glucoamylase activity provided by non-natural yeast over the reference yeast in an amount in the range of about 1.15 to about 7.5 times greater, about 1.175 to about 7.5 times greater, about 1.225 to about 7.5 times greater, about 1.25 to about 7.5 times greater, about 1.3 to about 7.5 times greater, about 1.35 to about 7.5 times greater, about 1.4 to about 7.5 times greater, about 1.45 to about 7.5 times greater, about 1.5 to about 7.5 times greater, about 1.75 to about 7.5 times greater, about 2 to about 7.5 times greater, about 2.25 to about 7.5 times greater, about 2.5 to about 7.5 times greater, about 2.75 to about 7.5 times greater, about 3 to about 7.5 times greater, about 3.25 to about 7.5 times greater, about 3.5 to about 7.5 times greater, about 3.75 to about 7.5 times greater, or about 4 to about 7.5 times greater in the non-natural yeast over the reference yeast.

Measurement of glucoamylase activity in the fermentation medium can be performed at a desired time point during fermentation. For example, a sample from the fermentation media can be taken about 1/10^(th), about 2/10^(th), about 3/10^(th), about 4/10^(th), about 5/10^(th), about 6/10^(th), about 7/10^(th), about 8/10^(th), about 9/10^(th) of the way through the fermentation process, or at the end of the fermentation process, and the sample can be tested for glucoamylase activity.

In some modes of practice, the fermentation period is about 30 hours or greater, about 40 hours or greater, about 50 hours or greater, or about 60 hours or greater, such as a period of time in the range of about 40 to about 120 hours, or 50 to about 110 hours.

The fermentation product (also referred to herein as a “bio-derived product” or “bioproduct”) can be any product that can be prepared by enzymatic degradation of a starch material by the glucoamylase, formation of glucose, and fermentation of glucose. In aspects, In an embodiment, the fermentation product is selected from the group consisting of: amino acids, organic acids, alcohols, diols, polyols, fatty acids, fatty acid alkyl esters (such as fatty acid methyl or ethyl esters (for example C6 to C12 fatty acid methyl esters (preferably C8 to C10 fatty acid methyl esters))), monacyl glycerides, diacyl glycerides, triacyl glycerides, and mixtures thereof. Preferred fermentation products are organic acids, amino acids, fatty acid alkyl esters (such as fatty acid methyl esters (for example C8 to C12 fatty acid methyl esters (preferably C8 to C10 fatty acid methyl esters))), and their salts thereof, and especially where the organic acid is selected from the group consisting of hydroxyl carboxylic acids (including mono-hydroxy and di-hydroxy mono-, di-, and tri-carboxylic acids), monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids and mixtures thereof. Examples of fermentation products that are prepared by the present process are organic acids or amino acids such as lactic acid, citric acid, malonic acid, hydroxy butyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid, 3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, acetic acid, methyl hexanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl dodecanoate, ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl dodecanoate, and mixtures thereof and derivatives thereof and salts thereof. In a preferred aspect, a fermentation method of the disclosure produces ethanol as the bioproduct.

The fermentation product is recovered from the fermentation broth. The manner of accomplishing this will depend on the particular product. However, in some modes of practice, the organism is separated from the liquid phase, typically via a filtration step or centrifugation step, and the product recovered via, for example, distillation, extraction, crystallization, membrane separation, osmosis, reverse osmosis, or other suitable technique.

The present process provides the ability to make fermentation products on a production scale level with excellent yields and purity. In an aspect, the process is carried out in fermentation broth quantities of at least 25,000 gallons. In an aspect, the batch process is carried out in to produce batches of at least 25,000 gallons of final fermentation broth. Add continuous process, vessels of at least 200,000 gallons

A composition comprising a Sc MFα2 SS or the Sc PHO5 SS—glucoamylase can optionally be used in combination with any one or in any combination with the following enzymes that are different than the glucoamylase. Exemplary other enzymes include alpha amylases, beta-amylases, peptidases (proteases, proteinases, endopeptidases, exopeptidases), pullulanases, isoamylases, cellulases, hemicellulases, endo-glucanases and related beta-glucan hydrolytic accessory enzymes, xylanases and xylanase accessory enzymes, acetolactate decarboxylases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolyzing enzymes and other glucoamylases.

In some aspects, a Sc MFα2 SS or the Sc PHO5 SS—glucoamylase can be used for starch conversion processes, such as for the production of dextrose for fructose syrups, specialty sugars and in alcohol and other end-product (e.g., organic acid, ascorbic acid, and amino acids). Production of alcohol from the fermentation of starch substrates using glucoamylases of the disclosure can include the production of fuel alcohol or potable alcohol.

The production of alcohol can be greater when a Sc MFα2 SS or the Sc PHO5 SS—glucoamylase of used under the same conditions as compared to the parent or wild-type glucoamylase. For example, the increase in alcohol production using the glucoamylases of the disclosure can be 1.1× or greater, 1.2× or greater, 1.3× or greater, 1.4× or greater, 1.5× or greater, 1.6× or greater, 1.7× or greater, 1.7× or greater, 1.8× or greater, 1.9× or greater, 2.0× or greater, 2.1× or greater, 2.2× or greater, 2.3× or greater, 2.4× or greater, or 2.5× or greater that alcohol production in a wild type strain.

In some aspects, the disclosure provides a method for producing ethanol by fermentation, wherein the ethanol is present in the fermentation medium at a concentration of 90 g/L or greater. In the method, a liquid medium comprising a starch material and a non-natural yeast comprising a exogenous nucleic acid encoding polypeptide comprising a glucoamylase portion and a signal sequence heterologous to the glucoamylase is fermented. Fermentation can provide an ethanol concentration of about 90 g/L or greater in the liquid medium, such as in the range of about 90 g/L to about 170 g/L, in the range of about 110 g/L to about 170 g/L, in the range of about 125 g/L to about 170 g/L, or in. in the range of about 140 g/L to about 170 g/L.

The method includes fermenting a liquid medium comprising a starch material and a non-natural yeast comprising a exogenous nucleic acid encoding polypeptide comprising a glucoamylase portion and a signal sequence heterologous to the glucoamylase, wherein said fermenting provides an ethanol concentration of 90 g/L or greater in the liquid medium.

Use of the non-natural yeast of the current disclosure may also provide benefits with regards to increased titers, reduced volatile organic acids (VOCs), and reduced fusel oil compounds (volatile organic acids, higher alcohols, aldehydes, ketones, fatty acids and esters).

The fermentation product may be first treated with one or more agents a treatment system. The treated fermentation product can then be sent to a distillation system. In the distillation system, the fermentation product can be distilled and dehydrated into ethanol. In some aspects, the components removed from the fermentation medium include water, soluble components, oil and unfermented solids. Some of these components can be used for other purposes, such as for an animal feed product. Other co-products, for example, syrup can be recovered from the stillage.

The present disclosure also provides a method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, such as wine, cider, vinegar, rice wine, soya sauce, or juice, said method comprising the step of treating a starch and/or sugar containing plant material with a composition as described herein. In another aspect, the invention also relates to a kit comprising a glucoamylase of the current disclosure, or a composition as contemplated herein; and instructions for use of said glucoamylase or composition. The invention also relates to a fermented beverage produced by a method using the glucoamylase.

After the fermentation process is complete, materials present in the fermentation medium can be of use. In some aspects, after a fermentation process has been completed, or while a fermentation process is ongoing, some or all of a bioproduct can be removed from the fermentation medium to provide a refined composition comprising non-bioproduct solids. The non-bioproduct solids the non-natural yeast, feedstock material in the medium that is not utilized by the yeast, as well as fermentation co-products. These materials can provide sources of carbohydrates and proteins that are useful as supplements to improve the nutritional content of a feed composition. The feed material can be a co-product from a fermentation process such as stillage (whole stillage, thin stillage, etc.) or composition prepared therefrom including dried distillers grains (DDG), distillers dry grains with solubles (DDGS), distillers wet grains (DWG), and distillers solubles (DS).

A fermentation medium, optionally with some or all of the target bioproduct removed, can be further treated, such as to remove water, or to cause precipitation or isolation of the non-bioproduct solids from the medium. In some cases the medium is treated by freeze drying or oven drying. After treatment the refined composition may be in the form of, for example, a liquid concentrate, a semi-wet cake, or a dry solid. The refined composition can be used as a feed composition itself, or an ingredient in the preparation of a feed composition. In preferred preparations, the feed composition is a livestock feed composition such as for sheep, cattle, pigs, etc.

The solids in the fermentation medium can provide a source of one or more amino acids. Introduced into an animal feed, the fermentation co-product can provide an enhanced amino acid content with regard to one or more essential amino acids. Essential amino acids can include histidine, isoleucine, lysine, methionine, phenylalanine, threonine, and tryptophan. These amino acids can be present in the feed composition as free amino acids or can be derived from proteins or peptides rich in the amino acids. The solids in the fermentation medium can provide a source of one prebiotics, which are nondigestible food substances, such as nondigestible oligosaccharides, that selectively stimulate the growth of favorable species of bacteria in the gut, thereby benefitting the host. The solids in the fermentation medium can provide a source of phytases, β-glucanases, proteases, and xylanases.

The feed composition can be used in aquaculture, is the farming of aquatic organisms such as fish, shellfish, or plants. Aquaculture includes the cultivation of both marine and freshwater species and can range from land-based to open-ocean production.

A feed composition, in addition to material obtained from the fermentation media, can include one or more feed additives. Feed additives can be used, for example, to help provide a balanced diet (e.g., vitamins and/or trace minerals), to protect the animals from disease and/or stress (e.g., antibiotics, probiotics) and/or to stimulate or control growth and behavior (e.g., hormones). Additive product ingredients may include, for example: growth promoters, medicinal substances, buffers, antioxidants, enzymes, preservatives, pellet-binding agents, direct-fed microbials, etc. Additive product ingredients may also include, for example, ionophores (e.g. monesin, lasalocid, laidlomycin, etc.), β-agonist (zilpaterol, ractompamine, etc.), antibiotics (e.g., chlortetracycline (CTC), oxytetracycline, bacitrain, tylosin, aureomycin), probiotics and yeast cultures, coccidiostats (e.g., amprollium, decoquinate, lasalocid, monensin), and hormones (e.g., growth hormones or hormones that inhibit estrus and/or ovulation such as melengestrol acetate), pheromones, nutraceuticals, pharmaceuticals, flavanoids, nutritive and non-nutritive supplements, detoxicants, etc. Some commercially available additives are sold under the trade names Rumensin®, Bovatec®, Deccox®, Tylan®, Optaflexx®, and MGA®.

EXAMPLE 1 Generation of Amylolytic Saccharomyces cerevisiae Strains

Strain 1-3: ura3Δ Saccharomyces cerevisiae Base Strain

Strain 1 is transformed with SEQ ID NO 1 (pAV18). SEQ ID NO 1 contains the following elements: i) an expression cassette for a mutant version of a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase gene from Saccharomyces cerevisiae (ARO4-OFP) corresponding to nucleotides 479-2647; ii) loxP sequence corresponding to nucleotides 445-478 and 2648-2681, and iii) flanking DNA for targeted chromosomal integration into integration locus A (URA3) where nucleotides 1-436 correspond to the URA3 5′ flanking region and nucleotides 2691-3182 correspond to the URA3 3′ flanking region. Transformants are selected on synthetic complete medium containing 3.5 g/L of p-fluorophenylalanine, and 1 g/L L-tyrosine (ScD-PFP). Resulting transformants are streaked for single colony isolation on ScD-PFP. A single colony is selected. Correct integration of SEQ ID NO: 1 into one allele of locus A is verified by PCR in the single colony. A PCR verified isolate is designated Strain 1-1.

Strain 1-1 is transformed with SEQ ID NO 2 (pCM520a). SEQ ID NO 2 contains the following elements: i) a codon optimized expression cassette for a acetamidase (amdS) gene from Aspergillus nidulans corresponding to nucleotides 638-2284 with a TEF1 promoter corresponding to nucleotides 2285-2740 and a TEF1 terminator corresponding to nucleotides 478-637; ii) loxP sequence corresponding to nucleotides 444-477 and 2741-2774, and iii) flanking DNA for targeted chromosomal integration into integration locus A (URA3) where nucleotides 1-435 correspond to the URA3 5′ flanking region and nucleotides 2783-3275 correspond to the URA3 3′ flanking region. Transformants are selected on Yeast Nitrogen Base (without ammonium sulfate or amino acids) containing 80 mg/L uracil and 1 g/L acetamide as the sole nitrogen source. Resulting transformants are streaked for single colony isolation on Yeast Nitrogen Base (without ammonium sulfate or amino acids) containing 80 mg/L uracil and 1 g/L acetamide as the sole nitrogen source. A single colony is selected. Correct integration of SEQ ID NO 2 into the second allele of locus A is verified by PCR in the single colony. A PCR verified isolate is designated Strain 1-2.

Strain 1-2 is co-transformed with SEQ ID NO 3 and SEQ ID NO 4. SEQ ID NO 3 contains the following elements: i) an open reading frame for a cre recombinase from P1 bacteriophage corresponding to nucleotides 53-1084, and ii) flanking DNA homologous to SEQ ID NO 4 corresponding to nucleotides 1-47 and 1086-1132. SEQ ID NO 4 contains the following elements: i) a 2μ origin of replication corresponding to nucleotides 2195-3350; ii) a URA3 selectable marker from Saccharomyces cerevisiae corresponding to nucleotides 3785-4901; and iii) flanking DNA containing a PGK promoter corresponding to nucleotides 5791-6376 and CYC1 terminator corresponding to nucleotides 10-199 from Saccharomyces cerevisiae. For the remaining part of SEQ ID NO 4, a pUC origin of replication corresponds to nucleotides 386-1053; and an ampicillin resistance gene corresponds to nucleotides 1204-2061. Transformants are selected on synthetic dropout medium lacking uracil (ScD-Ura). ScD-Ura agar plates contain: 20 g/L agar, 6.7 g/L Yeast Nitrogen Base, 2 g Synthetic complete drop-out mix lacking uracil, and 20 g/L dextrose. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. The isolated colony is screened for growth on ScD-PFP and Yeast Nitrogen Base (without ammonium sulfate or amino acids) containing 80 mg/L uracil and 1 g/L acetamide as the sole nitrogen source. Loss of the ARO4-OFP and amdS genes is verified by PCR. The PCR verified isolate is streaked to YNB containing 5-FOA to select for loss of the 2μ plasmid. The PCR verified isolate is designated Strain 1-3.

Strain 1-4: Saccharomyces cerevisiae Expressing Saccharomycopsis fibuligera Glucoamylase

Strain 1-3 is transformed with SEQ ID NO 5. SEQ ID NO 5 contains the following elements: 1) an expression cassette for a glucoamylase gene from Saccharomycopsis fibuligera corresponding to nucleotides 2769-4316, including an ADH1 promoter corresponding to nucleotides 2022-2768 and a CYC1 terminator corresponding to nucleotides 4318-4540, 2) a centromere to allow for stable replication corresponding to nucleotides 6798-7316, and 3) an expression cassette for an orotidine-5′-phosphate decarboxylase (URA3) corresponding to nucleotides 195-1292. Transformants are selected on ScD-Ura. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-4a, b, c,

Strain 1-5: Saccharomyces cerevisiae Expressing a Modified Saccharomycopsis fibuligera Glucoamylase Containing an N-terminal Secretion Leader from Alpha Mating Factor 1 (MFα1)

Strain 1-3 is transformed with SEQ ID NO 6. SEQ ID NO 6 contains the following elements: 1) an expression cassette for a modified glucoamylase gene from Saccharomycopsis fibuligera containing an N-terminal secretion leader from alpha mating factor 1 (MFα1) corresponding to nucleotides 2769-4319, including an ADH1 promoter corresponding to nucleotides 2023-2768 and a CYC1 terminator corresponding to nucleotides 4320-4543, 2) a centromere to allow for stable replication (CEN6) corresponding to nucleotides 6801-7319, and 3) an expression cassette for an orotidine-5′-phosphate decarboxylase (URA3) corresponding to nucleotides 195-1292. SEQ ID NO 6 also includes an ampicillin resistance gene corresponding to nucleotides 5809-6669. Transformants are selected on ScD-Ura. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-5a, b, c.

Strain 1-6: Saccharomyces cerevisiae Expressing a Modified Saccharomycopsis fibuligera Glucoamylase Containing an N-terminal Secretion Leader from Alpha Mating Factor 2(MFα2)

Strain 1-3 is transformed with SEQ ID NO 7. SEQ ID NO 7 contains the following elements: 1) an expression cassette for a modified glucoamylase gene from Saccharomycopsis fibuligera containing an N-terminal secretion leader from alpha mating factor 2 (MFα2) corresponding to nucleotides 2769-4319, including an ADH1 promoter corresponding to nucleotides 2022-2768 and a CYC1 terminator corresponding to nucleotides 4320-4543, 2) a centromere to allow for stable replication (CEN6) corresponding to nucleotides 6801-7319, and 3) an expression cassette for an orotidine-5′-phosphate decarboxylase (URA3) corresponding to nucleotides 195-1229 SEQ ID NO 7 also includes an ampicillin resistance gene corresponding to nucleotides 5809-6669. Transformants are selected on ScD-Ura. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-6 a, b, c.

Strain 1-7: Saccharomyces cerevisiae Expressing a Modified Saccharomycopsis fibuligera Glucoamylase Containing an N-terminal Secretion Leader from Acid Phosphatase (PHO5)

Strain 1-3 is transformed with SEQ ID NO 8. SEQ ID NO 8 contains the following elements: 1) an expression cassette for a modified glucoamylase gene from Saccharomycopsis fibuligera containing an N-terminal secretion leader from Saccharomyces cerevisiae acid phosphatase (PHO5) corresponding to nucleotides 2769-4313, including an ADH1 promoter corresponding to nucleotides 2023-2768 and a CYC1 terminator corresponding to nucleotides 4314-4537, 2) a centromere to allow for stable replication (CEN6) corresponding to nucleotides 6795-7313, and 3) an expression cassette for an orotidine-5′-phosphate decarboxylase (URA3) corresponding to nucleotides 195-1292. SEQ ID NO 8 also includes an ampicillin resistance gene corresponding to nucleotides 5803-6663. Transformants are selected on ScD-Ura. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-7 a, b, c.

Strain 1-8: Saccharomyces cerevisiae Plasmid Control

Strain 1-3 is transformed with SEQ ID NO 9. SEQ ID NO 9 contains the same elements as SEQ ID NO 5-8, with the exception that it lacks an expression cassette for a glucoamylase. Transformants are selected on ScD-Ura. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-8 a, b, c

Strain 1-9: Saccharomyces cerevisiae Expressing a Modified Saccharomycopsis fibuligera Glucoamylase Containing an N-terminal Secretion Leader from a Mating Factor 1 (MFα1)

Strain 1-3 is transformed with SEQ ID NO 15. SEQ ID NO 15 contains the following elements: 1) an expression cassette for a modified glucoamylase gene from Saccharomycopsis fibuligera containing an N-terminal secretion leader from a mating factor 1 (MFα1) corresponding to nucleotides 2769-4337, including an ADH1 promoter corresponding to nucleotides 2023-2768 and a CYC1 terminator corresponding to nucleotides 4338-4561, 2) a centromere to allow for stable replication (CEN6) corresponding to nucleotides 6819-7337, and 3) an expression cassette for an orotidine-5′-phosphate decarboxylase (URA3) corresponding to nucleotides 195-1292. SEQ ID NO 15 also includes an ampicillin resistance gene corresponding to nucleotides 5827-6687. Resulting transformants are streaked for single colony isolation on ScD-Ura. A single colony is selected. Presence of the plasmid is verified by PCR. The PCR verified isolate is designated Strain 1-9 a, b, c.

TABLE 3 Description of Engineered Yeast Strain Description Parent Strain Saccharomyces cerevisiae N/A 1 (Lasqffre, Ethanol Red) Strain ura3Δ/URA3, ARO4-OFP+ Strain 1-1 1 Strain ura3Δ, ARO4-OFP+, amdS+ Strain 1-2 1-1 Strain ura3Δ Strain 1-3 1-2 Strain Saccharomycopsis fibuligera Strain 1-4 a, b, c GLA1+ 1-3 Strain Saccharomycopsis fibuligera Strain 1-5 a, b, c GLA1+ (w/MFα1 leader) 1-3 Strain Saccharomycopsis fibuligera Strain 1-6 a, b, c GLA1+ (w/MFα2 leader) 1-3 Strain Saccharomycopsis fibuligera Strain 1-7 a, b, c GLA1+ (w/PHO5 leader) 1-3 Strain Vector control Strain 1-8 a, b, c 1-3 Strain Saccharomycopsis fibuligera Strain 1-9 a, b, c GLA+ (w/MFa1 leader) 1-3

EXAMPLE 2 Evaluation of Amylolytic Saccharomyces cerevisaie Strains in Simultaneous Saccharification and Fermentation Shake Flask Assays Shake Flask Evaluation Using Partially Hydrolyzed Corn Starch (AV_2014-08-20, Changing the Secretion Signal of the Sf GA)

A subset of strains listed in Table 3 are streaked out on a ScD-Ura plate and incubated at 30° C. until single colonies are visible (1-2 days). Cells from the ScD-Ura plate are scraped into sterile shake flask medium and the optical density (OD₆₀₀) is measured. Optical density is measured at wavelength of 600 nm with a 1 cm path length using a model Genesys20 spectrophotometer (Thermo Scientific).

A shake flask is inoculated with the cell slurry to reach an initial OD₆₀₀ of 0.1-0.3. Immediately prior to inoculating, 50 mL of shake flask medium is added to a 250 mL non-baffled shake flask (Coming 4995-250) fitted with a screw cap containing a gas-permeable seal (corning 1395-45LTMC) The shake flask medium consists of 850 g partially hydrolyzed corn starch (% DS 30-37, DE 5-15), 150 g filtered light steep water, 25 g glucose, and 1 g urea (Sigma U6504).

The inoculated flask is incubated at 30° C. with shaking in an orbital shake at 100 rpm for 72 hours. Samples are taken and analyzed for ethanol concentrations in the broth during fermentation by high performance liquid chromatography with refractive index detector.

The results of the shake flask, shown in Table 4, demonstrate an improvement in ethanol titer in strains expressing either the MFα2 or PHO5 leader sequence on the Saccharomycopsis fibuligera glucoamylase relative to strains expressing the native Saccharomycopsis fibuligera glucoamylase.

TABLE 4 Ethanol titers for strains expressing the Saccharomycopsis fibuligera glucoamylase with and without altered leader sequence. Ethanol Titer at 72 Hours Strain Description (g/L) Strain Saccharomycopsis fibuligera 83.6, 89.2, 81.6 1-4a, b, c GLA1+ Strain Saccharomycopsis fibuligera 25.6, 25.5, 25.1 1-5a GLA1+ (w/MFα1 leader) Strain Saccharomycopsis fibuligera 156.3, 158.7, 158.5 1-6a, b, c GLA1+ (w/MFα2 leader) Strain Saccharomycopsis fibuligera 156.7, 156.8, 157.1 1-7a, b, c GLA1+ (w/PHO5 leader) Strain Vector control 25.6, 25.8, 25.6 1-8a, b, c Strain Saccharomycopsis fibuligera 26.4, 26.5, 26.3 1-9a, b, c GLA1+ (w/MFa1 leader)

EXAMPLE 3 Enzyme Production by Amylolytic Saccharomyces cerevisaie Strains Shake Flask Evaluation Using Raffinose Grown Cells to Evaluate Enzyme Production

A subset of strains listed in Table 3 are streaked out on a ScD-Ura plate and incubated at 30° C. until single colonies are visible (1-2 days). Cells from the ScD-Ura plate are scraped into sterile shake flask medium and the optical density (OD₆₀₀) is measured. Optical density is measured at wavelength of 600 nm with a 1 cm path length using a model Genesys20 spectrophotometer (Thermo Scientific).

A shake flask is inoculated with the cell slurry to reach an initial OD₆₀₀ of 0.1-0.3. Immediately prior to inoculating, 100 mL of shake flask medium is added to a 500 mL baffled shake flask. The shake flask medium consists of 5% Raffinose (Sigma R0250), 6.7 g Yeast Nitrogen Base (Difco 291940), and 1.9 g synthetic complete amino acid dropout mix (MP Biomedicals 4410-622).

The inoculated flask is incubated at 30° C. with shaking in an orbital shake at 250 rpm for 24 hours. Samples are taken and analyzed for OD₆₀₀ and cell dry weight. Cells were removed by centrifugation and 90 mls of broth was concentrated to 1.0 ml using a 10K MWCO column (Millipore UFC901024) and frozen prior to analysis.

Glucoamylase activity was measured in the concentrated broth by coupling starch hydrolysis to HXK/G6PDH reactions in a two-step end point assay (Sigma G3293). A unit (U) of GA activity can be defined as the amount of enzyme that catalyzes the release of 1 mg glucose/min from starch. Glucoamylase activity can be measured in concentrated broth by coupling starch hydrolysis to a HXK/G6PDH reaction mix (Sigma G3293) in a two-step end point assay. Broth can be concentrated from a predetermined amount of cells grown using a non-glucose carbon source (i.e. raffinose) to avoid interference with the assay.

The specific activity is equal to the activity in a given volume of broth divided by the wet weight of cells in the same volume of broth. Specific activity has the following units, U of GA activity per gram of biomass (U/g biomass). The amount of biomass used in the assay can be measured by determining the wet cell weight after removing the broth, either by filtration or centrifugation.

A starch solution is prepared by dissolving 1.1 g of corn starch (S4126, Sigma) in 50 mL of near boiling water, then adding 1 mL of 3M sodium acetate pH 5.2. A volume of concentrated broth (V_(b)), typically in the range of 1-20 ul (prepared by using a 10 Kb Kd cutoff column, Millipore #UFC901008) is added to the starch slurry (V_(s)), in a total volume of 200 ul, and allowed to incubate at 37° C. for a specific period of time (T), typically between 5-60 minutes. Parameters are selected such that the glucose formation is linear within a desired time. 20 μL of each sample is added to 2 μL 0.6N NaOH and mixed well. 200 μL of the HXK/G6PDH mix is then added and incubated at 30° C. for 30 minutes. The absorbance at 340 nm is measured using a spectrophotometer (SpectraMax™ M2). Regression analysis using known glucose standards is used to calculate the amount of glucose released in each sample. The specific enzyme activity per gram of biomass (U/g biomass) can be calculated by obtaining the weight in grams of the sample used prior to concentration. Unit of activity=(mg glucose/T)*((V_(b)+V_(s))/(V_(b)))*(222/20). Specific activity=Unit of activity/g biomass.

Protein concentrations were determined using Advanced Protein Assay Reagent (Cytoskeleton ADV01). A standard denaturing SDS-PAGE protein gel was run using 16 μl of concentrated broth combined with 4 μl of loading buffer from strains 1-4a 1-4b, 1-6a, 1-6b, 1-7a, 1-7b, 1-8a and 1-8b.

The results of the shake flask, shown in Table 5 and FIG. 1, demonstrate the beneficial effect of the MFα2 and the PHO5 leader sequence on Saccharomycopsis fibuligera glucoamylase secretion and activity.

TABLE 5 Enzyme production for strains expressing the Saccharomycopsis fibuligera glucoamylase with and without altered leader sequence. Extracellular Extracellular Enzyme activity protein Strain Description (U/g biomass) (mg/mL) Strain Saccharomycopsis fibuligera 2.06, 1.91 0.06, 0.06 1-4a, b GLA1+ Strain Saccharomycopsis fibuligera n.d. n.d. 1-5a, b GLA1+ (w/MFα1 leader) Strain Saccharomycopsis fibuligera 7.33, 8.24 0.15, 0.21 1-6a, b GLA1+ (w/MFα2 leader) Strain Saccharomycopsis fibuligera 7.96, 10.5 0.21, 0.19 1-7a, b GLA1+ (w/PHO5 leader) Strain Vector control −0.04, −0.01 0.02, 0.07 1-8a, b 

1. A polypeptide comprising (a) a secretion signal amino acid sequence having 90% or greater identity to SEQ ID NO:10 or SEQ ID NO:11 and (b) a glucoamylase amino acid sequence from a yeast, fungal, or bacterial glucoamylase polypeptide, wherein the polypeptide has glucoamylase activity.
 2. The polypeptide of claim 1, wherein the glucoamylase amino acid sequence in (b) is a glucoamylase amino acid sequence having 90% or greater sequence identity to amino acids 19-515 of SEQ ID NO:12.
 3. The polypeptide of claim 1 having 95% or greater sequence identity to SEQ ID NO:13.
 4. The polypeptide of claim 3 comprising SEQ ID NO:
 13. 5. The polypeptide of claim 1 having 95% or greater sequence identity to SEQ ID NO:14.
 6. The polypeptide of claim 5 comprising SEQ ID NO:14.
 7. The polypeptide of claim 1 further comprising a third sequence that is different than SEQ ID NO:10, SEQ ID NO:11, or the glucoamylase amino acid sequence, wherein the third sequence is positioned between SEQ ID NO:10 and the glucoamylase amino acid sequence, or SEQ ID NO:11 and the glucoamylase amino acid sequence.
 8. The polypeptide of claim 1 wherein the glucoamylase amino acid sequence is from a yeast or fungal glucoamylase.
 9. The polypeptide of claim 8 wherein the glucoamylase amino acid sequence is from a yeast or fungal organism selected from the group consisting of Amorphotheca resinae, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus kawachii, Aspergillus shirousami, Blastobotrys adeninivorans, Candida albicans, Rhizopus oryzae, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Brettanomyces bruxellensis, and Cyberlindnera jadinii.
 10. The polypeptide of claim 1 wherein the glucoamylase amino acid sequence is an enzymatically active portion of a yeast or fungal glucoamylase polypeptide.
 11. The polypeptide of claim 2 wherein the glucoamylase amino acid sequence has 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to amino acids 19-515 of SEQ ID NO:12.
 12. The polypeptide of claim 11 wherein the glucoamylase amino acid sequence has 100% identity to amino acids 19-515 of SEQ ID NO:12. 13-19. (canceled)
 20. A host cell that expresses the polypeptide of claim
 1. 21-22. (canceled)
 23. The host cell of claim 20 wherein the host cell is a strain of Saccharomyces cerevisiae.
 24. The host cell of claim 23 which is (a) tolerant to growth in fermentation medium having a concentration of ethanol of greater than 90 g/L, (b) tolerant to growth in at temperatures of greater than 33° C., such as in the range of 34° C.-40° C., or both (a) and (b). 25-37. (canceled)
 38. A fermentation method for producing ethanol to a concentration of 90 g/L or greater in a medium, comprising a step of: fermenting a liquid medium comprising a starch material and a non-natural yeast comprising an exogenous nucleic acid encoding a polypeptide comprising a glucoamylase portion and a signal sequence heterologous to the glucoamylase, wherein said fermenting provides an ethanol concentration of 90 g/L or greater in the liquid medium. 39-42. (canceled)
 43. The method of claim 38 wherein said signal sequence comprises SEQ ID NO: 13 or SEQ ID NO: 